The K562 erythroleukemia cell line constitutively expresses low levels of embryonic and fetal but not adult hemoglobin and can be further induced by chemical stimuli for hemoglobin production. Within the cell, regulation of transcriptional activity of globin genes is determined by a variety of cis- acting regulatory DNA sequences and trans-acting proteins or other factors. Several cis-acting sequences have been identified through naturally occurring and laboratory synthesized mutations or deletions. One of these is the silencer located in the region between about -250 and -300 5' in the epsilon-globin gene which has been identified by deletion mutations in transient transfection assays. To examine in detail the molecular mechanism of the epsilon-globin gene silencer activity and its possible role in the developmental regulation of epsilon-globin gene expression, further characterization of the epsilon- globin gene silencer is necessary. In particular, determination of the silencer activity in non-deleted or non-truncated mutations of the epsilon- globin gene is essential. We have mutated six nucleotides in the silencer region of an epsilon-globin gene containing 1.4kb of 5' flanking sequence. The activity of this 1.4kb 5' flanking sequence fused to a reporter gene (luciferase) was 3 to 6 fold greater than the wild type sequence when assayed for transient expression in K562 cells. This study confirms the importance of the epsilon-globin gene silencer and supports its possible role in regulation of developmental expression. The deletion and site directed mutations of the epsilon-globin gene will be examined for their ability to correctly initiate transcription as a possible mechanism of action for the silencer.